Mycobacterium avium paratuberculosis invades human small-intestinal goblet cells and elicits inflammation.

Crohn disease is a chronic inflammatory bowel disease of unknown etiology. Mycobacterium avium paratuberculosis (MAP) was found in the gut of patients with Crohn disease, but causality was not established. Fully developed, germ-free human small intestine and colon were established by subcutaneous transplantation of fetal gut into SCID (severe combined immunodeficiency) mice thereafter infected by direct intraluminal inoculation of MAP. We have found that MAP actively invades the human gut epithelial goblet cells of the small intestine, inducing severe tissue damage and inflammation. These observations indicate that MAP can specifically colonize the normal human small intestine and can elicit inflammation and severe mucosal damage.

Role of type III secretion in Edwardsiella tarda virulence.

Edwardsiella tarda is a Gram-negative enteric bacterium affecting both animals and humans. Recently, a type III secretion system (TTSS) was found in Ed. tarda. Such systems are generally used by bacterial pathogens to deliver virulence factors into host cells to subvert normal cell functions. Genome-walking was performed from the eseB and esrB genes (homologues of Salmonella sseB and ssrB, respectively) identified in previous studies, to determine the sequences of the TTSS. Thirty-five ORFs were identified which encode the TTSS apparatus, chaperones, effectors and regulators. Mutants affected in genes representing each category were generated and found to have decreased survival and growth in fish phagocytes. LD(50) values of the mutants were increased by at least 10-fold in comparison to those of the wild-type strain. The adherence and invasion rates of the esrA and esrB mutants were enhanced while those of the other mutants remained similar to the wild-type. The eseC and eseD mutants showed slight autoaggregation in Dulbecco's Modified Eagle Medium, whereas the rest of the mutants failed to autoaggregate. Regulation of the TTSS was found to involve the two-component regulatory system esrA-esrB. This study showed that the TTSS is important for Ed. tarda pathogenesis. An understanding of this system will provide greater insight into the virulence mechanisms of this bacterial pathogen.

Comparative proteomic analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli strains and their ihf and ler mutants.

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens that are responsible for food-borne epidemics in many countries. Integration host factor (IHF) and the locus of enterocyte effacement-encoded regulator (Ler) are needed for the expression of virulence genes in EHEC and EPEC, including the elicitation of actin rearrangements for attaching and effacing lesions. We applied a proteomic approach, using two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry and a protein database search, to analyze the extracellular protein profiles of EHEC EDL933, EPEC E2348/69, and their ihf and ler mutants. Fifty-nine major protein spots from the extracellular proteomes were identified, including six proteins of unknown function. Twenty-six of them were conserved between EHEC EDL933 and EPEC E2348/69, while some of them were strain-specific proteins. Four common extracellular proteins (EspA, EspB, EspD, and Tir) were regulated by both IHF and Ler in EHEC EDL933 and EPEC E2348/69. TagA in EHEC EDL933 and EspC and EspF in EPEC E2348/69 were present in the wild-type strains but absent from their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of EPEC E2348/69. Two dominant forms of EspB were found in EHEC EDL933 and EPEC E2348/69, but the significance of this is unknown. These results show that proteomics is a powerful platform technology for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnoses of these pathogens.

The filamentous type III secretion translocon of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (TTSS) to inject effector proteins into the plasma membrane and cytosol of infected cells. To translocate proteins, EPEC, like Salmonella and Shigella, is believed to assemble a macromolecular complex (type III secreton) that spans both bacterial membranes and has a short needle-like projection. However, there is a special interest in studying the EPEC TTSS owing to the fact that one of the secreted proteins, EspA, is assembled into a unique filamentous structure also required for protein translocation. In this report we present electron micrographs of EspA filaments which reveal a regular segmented substructure. Recently we have shown that deletion of the putative structural needle protein, EscF, abolished protein secretion and formation of EspA filaments. Moreover, we demonstrated that EspA can bind directly to EscF, suggesting that EspA filaments are physically linked to the EPEC needle complex. In this paper we provide direct evidence for the association between an EPEC bacterial membrane needle complex and EspA filaments, defining a new class of filamentous TTSS.

Construction of mini-Tn5cyaA' and its utilization for the identification of genes encoding surface-exposed and secreted proteins in Bordetella bronchiseptica.

A mini-Tn5 transposon derivative, mini-Tn5cyaA', has been constructed. It contains a promoter-less and ribosome binding site-deficient reporter gene, encoding the catalytic domain of Bordetella pertussis adenylate cyclase toxin (CyaA'). We used this system to mutagenize B. bronchiseptica and we developed a screen for identification of mutants containing cyaA' translational fusions. This system was used to identify B. bronchiseptica genes that encode surface-exposed and secreted proteins.

De novo formation of focal complex-like structures in host cells by invading Streptococci.

Group A streptococcus (GAS) induces its own entry into eukaryotic cells in vitro and in vivo. Fibronectin (Fn) bound to protein F1, a GAS surface protein, acts as a bridge connecting the bacterium to host cell integrins. This triggers clustering of integrins, which acquire a polar pattern of distribution similar to that of protein F1 on the GAS surface. A unique and transient adhesion complex is formed at the site of GAS entry, which does not contain alpha-actinin. Vinculin is recruited to the site of GAS entry but is not required for uptake. The invading GAS recruits focal adhesion kinase (FAK), which is required for uptake and is tyrosine phosphorylated. The Src kinases, Src, Yes and Fyn, enhance the efficiency of GAS uptake but are not absolutely required for GAS entry. In addition, Rac and Cdc42, but not Rho, are required for the entry process. We suggest a model in which integrin engagement by Fn-occupied protein F1 triggers two independent signalling pathways. One is initiated by FAK recruitment and tyrosine phosphorylation, whereas the other is initiated by the recruitment and activation of Rac. The two pathways subsequently converge to trigger actin rearrangement leading to bacterial uptake.

Tir tyrosine phosphorylation and pedestal formation are delayed in enteropathogenic Escherichia coli sepZ::TnphoA mutant 30-5-1(3).

Enteropathogenic Escherichia coli (EPEC) strain 30-5-1(3) has been reported to form attaching and effacing (A/E) lesions without Tir tyrosine phosphorylation. In this study, we show that 30-5-1(3), which has a transposon insertion within the sepZ gene, forms wild-type A/E lesions including Tir tyrosine phosphorylation, but at a slower rate. A/E lesion formation by 30-5-1(3) occurs without detectable secretion of Tir or other EPEC Esp secreted proteins.

Characterization of Escherichia coli DNA lesions generated within J774 macrophages.

Macrophages are armed with multiple oxygen-dependent and -independent bactericidal properties. However, the respiratory burst, generating reactive oxygen species, is believed to be a major cause of bacterial killing. We exploited the susceptibility of Escherichia coli in macrophages to characterize the effects of the respiratory burst on intracellular bacteria. We show that E. coli strains recovered from J774 macrophages exhibit high rates of mutations. We report that the DNA damage generated inside macrophages includes DNA strand breaks and the modification 8-oxo-2'-deoxyguanosine, which are typical oxidative lesions. Interestingly, we found that under these conditions, early in the infection the majority of E. coli cells are viable but gene expression is inhibited. Our findings demonstrate that macrophages can cause severe DNA damage to intracellular bacteria. Our results also suggest that protection against the macrophage-induced DNA damage is an important component of the bacterial defense mechanism within macrophages.

Hierarchy in the expression of the locus of enterocyte effacement genes of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) elicit changes in host cell morphology and cause actin rearrangement, a phenotype that has commonly been referred to as attaching/effacing (AE) lesions. The ability of EPEC to induce AE lesions is dependent upon a type III protein secretion/translocation system that is encoded by genes clustered in a 35.6 kb DNA segment, named the locus of enterocyte effacement (LEE). We used transcriptional fusions between the green fluorescent protein (gfp) reporter gene and LEE genes rorf2, orf3, orf5, escJ, escV and eae, together with immunoblot analysis with antibodies against Tir, intimin, EspB and EspF, to analyse the genetic regulation of the LEE. The expression of all these LEE genes was strictly dependent upon the presence of a functional integration host factor (IHF). IHF binds specifically upstream from the ler (orf1) promoter and appears to activate expression of ler, orf3, orf5 and rorf2 directly. The ler-encoded Ler protein was involved in activating the expression of escJ, escV, tir, eae, espB and espF. Expression of both IHF and Ler was needed to elicit actin rearrangement associated with AE lesions. In conclusion, IHF directly activates the expression of the ler and rorf2 transcriptional units, and Ler in turn mediates the expression of the other LEE genes.

Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide.

In eukaryotes, tyrosine protein phosphorylation has been studied extensively, while in bacteria, it is considered rare and is poorly defined. We demonstrate that Escherichia coli possesses a gene, etk, encoding an inner membrane protein that catalyses tyrosine autophosphorylation and phosphorylation of a synthetic co-polymer poly(Glu:Tyr). This protein tyrosine kinase (PTK) was termed Ep85 or Etk. All the E.coli strains examined possessed etk; however, only a subset of pathogenic strains expressed it. Etk is homologous to several bacterial proteins including the Ptk protein of Acinetobacter johnsonii, which is the only other known prokaryotic PTK. Other Etk homologues are AmsA of the plant pathogen Erwinia amylovora and Orf6 of the human pathogen Klebsiella pneumoniae. These proteins are involved in the production of exopolysaccharide (EPS) required for virulence. We demonstrated that like Etk, AmsA and probably also Orf6 are PTKs. Taken together, these findings suggest that tyrosine protein phosphorylation in prokaryotes is more common than was appreciated previously, and that Etk and its homologues define a distinct protein family of prokaryotic membrane-associated PTKs involved in EPS production and virulence. These prokaryotic PTKs may serve as a new target for the development of new antibiotics.

Roles of integrins and fibronectin in the entry of Streptococcus pyogenes into cells via protein F1.

Entry of group A streptococcus (GAS) into cells has been suggested as an important trait in GAS pathogenicity. Protein F1, a fibronectin (Fn) binding protein, mediates GAS adherence to cells and the extracellular matrix, and efficient cell internalization. We demonstrate that the cellular receptors responsible for protein F1-mediated internalization of GAS are integrins capable of Fn binding. In HeLa cells, bacterial entry is blocked by anti-beta1 integrin monoclonal antibody. In the mouse cell line GD25, a beta1 null mutant, the alphavbeta3 integrin promotes GAS entry. Internalization of these cells by GAS is blocked by a peptide that specifically binds to alphavbeta3 integrin. In both cell lines, entry of GAS requires the occupancy of protein F1 by Fn. Neither the 29 kDa nor the 70 kDa N-terminal fragments or the 120 kDa cell-binding fragment of Fn promote bacterial entry. Fn-coated beads are taken up efficiently by HeLa cells. Both the entry of GAS via protein F1 and the uptake of Fn-coated beads are blocked by anti-beta1 antibody but are unaffected by a large excess of soluble Fn. Internalization of HeLa cells by bacteria bearing increasing amounts of prebound Fn to protein F1 reveals a sigmoidal ultrasensitive curve. These suggest that the ability of particles to interact via Fn with multiple integrin sites plays a central role in their ability to enter cells.

Interaction of enteropathogenic Escherichia coli with host epithelial cells.

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. Upon infection, EPEC induces the assembly of highly organized pedestal-like actin structures in host epithelial cells. All the EPEC genes that are involved in inducing formation of actin pedestals are located in a unique 35 kbp chromosomal pathogenicity island, termed LEE. These genes include the sep genes that encode components of type III protein secretion system, and genes that encode proteins secreted by this system, the esp genes. This protein secretion system is activated upon contact with the host cell, resulting in increased secretion of Esp proteins. Some of these Esp proteins from the translocation apparatus while others are translocated into the cytoplasm of the host cell. Concerted activity of the LEE genes including the eae, esp and the sep genes is needed to trigger signal transduction in the host cell which results in formation of an actin pedestal.

A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells.

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, employ a type III secretion system to deliver effector proteins across the bacterial cell. In EPEC, four proteins are known to be exported by a type III secretion system_EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor (Tir) protein (formerly Hp90) which is tyrosine-phosphorylated following transfer to the host cell to become a receptor for intimin-mediated intimate attachment and 'attaching and effacing' (A/E) lesion formation. The structural basis for protein translocation has yet to be fully elucidated for any type III secretion system. Here, we describe a novel EspA-containing filamentous organelle that is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells.

Protein translocation into host epithelial cells by infecting enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in young children. EPEC induces the formation of actin pedestal in infected epithelial cells. A type III protein secretion system and several proteins that are secreted by this system, including EspB, are involved in inducing the formation of the actin pedestals. We have demonstrated that contact of EPEC with HeLa cells is associated with the induction of production and secretion of EspB. Shortly after infection, EPEC initiates translocation of EspB, and EspB fused to the CyaA reporter protein (EspB-CyaA), into the host cell. The translocated EspB was distributed between the membrane and the cytoplasm of the host cell. Translocation was strongly promoted by attachment of EPEC to the host cell, and both attachment factors of EPEC, intimin and the bundle-forming pili, were needed for full translocation efficiency. Translocation and secretion of EspB and EspB-CyaA were abolished in mutants deficient in components of the type III protein secretion system, including sepA and sepB mutants. EspB-CyaA was secreted but not translocated by an espB mutant. These results indicate that EspB is both translocated and required for protein translocation by EPEC.

Agents that inhibit Rho, Rac, and Cdc42 do not block formation of actin pedestals in HeLa cells infected with enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.

Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements.

Enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide. Both pathogens colonize the intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the 'attaching and effacing' (A/E) lesion. Although EPEC was the first E. coli to be associated with human disease in the 1940s and 1950s, it was not until the late 1980s and early 1990s that the mechanisms and bacterial gene products used to induce this complex brush border membrane lesion and diarrhoeal disease started to be unravelled. During the past few months, there has been a burst of new data that have revolutionized some basic concepts of the molecular basis of bacterial pathogenesis in general and EPEC pathogenesis in particular. Major breakthroughs and developments in the genetic basis of A/E lesion formation, signal transduction, protein translocation, host cell receptors and intestinal colonization are highlighted in this review.

Enteropathogenic E. coli exploitation of host epithelial cells.

Enteropathogenic E. coli (EPEC) is a leading cause of neonatal diarrhea worldwide. These organisms adhere to the intestinal cell surface, causing rearrangement in the epithelial cell surface and underlying cytoskeleton, resulting in a structure termed an attaching/effacing (A/E) lesion. A/E lesion formation is thought necessary for EPEC-mediated disease. EPEC secretes several proteins that trigger signal transduction, intimate adherence, and cytoskeletal rearrangements in epithelial cells. Additionally, it produces intimin, an outer membrane product that mediates intimate adherence. Together these various bacterial molecules contribute to the intimate relationship that is formed by EPEC with host epithelial cells which results in A/E lesion formation and diarrhea.

Listeriolysin O activates mitogen-activated protein kinase in eucaryotic cells.

Infection with Listeria monocytogenes induces the activation of mitogen-activated protein (MAP) kinase in several tissue culture cell lines (P.Tang, I. Rosenshine, and B. B. Finlay, Mol. Biol. Cell 5:455-464, 1994). After various mutants were examined, the bacterial factor responsible for MAP kinase activation was identified as listeriolysin O (LLO). Growth supernatant containing LLO or purified LLO alone can induce MAP kinase tyrosine phosphorylation in HeLa cells. Single-amino-acid mutations in LLO that do not affect its membrane binding capacity but reduce its cytolytic activity also reduced its ability to induce MAP kinase activity in HeLa cells. Streptolysin O, another sulfhydryl-activated hemolysin, and the detergent saponin are also able to activate MAP kinase in target cells. Thus, the increased MAP kinase activity observed in L. monocytogenes-infected cells is most likely a result of the permeabilization of the host cell membrane by LLO and may not be linked with invasion.